Not applicable.
The invention relates to blood treating material having the capability of selectively removing endotoxin and cytokine inducing substances from blood or plasma by extracorporeal adsorption for therapeutic septic shock treatment.
Endotoxins are lipopolysaccharides from gram-negative bacteria and are the leading cause of sepsis and septic shock, having mortality rates of more than 50%. Endotoxins can persist in blood subsequent to infection even in the absence of live bacteria. Endotoxin molecules have a highly conserved region, which consists of Lipid-A moiety comprising several long fatty acid chains and sugar rings with at least two negatively charged phosphate groups. The Lipid-A moiety is connected to polysaccharide chains which vary greatly depending on bacteria type. The pathological effect is mainly derived from the Lipid-A moiety of the molecule. The accessibility of the Lipid-A moiety is largely modulated by the nature of the polysaccharide chains and the surrounding media, including such factors as salinity, water, sugar molecules, plasma, blood, pH, detergents, and the like. For example, high salt concentration leads to micellar and other supermolecular structures of endotoxins, resulting in different activities.
Endotoxin is assayed by measuring its cytokine-inducing effect on CD14-positive leucocytes, to produce, e.g. TNF-xcex1, which can be analyzed by an ELISA technique using a commercially available kit, e.g. RandD Systems, Bad Homburg, Germany or by an LAL induced chromogenic substrate reaction (Chromogenics, Moeldwagen, Sweden). The molecular weight of endotoxin ranges from 5000 Da to some millions Da depending on the polysaccharide chain length and its supermolecular structure.
Most extracorporeal removal strategies have exploited the negatively charged phosphate groups of endotoxin using positively charged adsorbent materials immobilized on a variety of substrates. Kodama, et al. (EP 0107119, EP 0129786) disclosed polycationic Polymyxin B covalently immobilized on polystyrene fibers and described adsorbing endotoxins from blood in a device filled with woven fibers of such material. Otto, et al. (EP 424698) also disclosed immobilized polycationic Polymyxin B on poly(comethacrylate) beads for adsorbing endotoxin from blood. Falkenhagen, et al. [Artificial Organs (1996) 20:420] described adsorbing endotoxin from plasma on polycationic polyethyleneimine-coated cellulosic beads. Mitzner, et al. [Artificial Organs (1993) 17(9):775] described polyethyleneimine and Polymyxin B immobilized on macroporous cellulose beads for endotoxin removal from plasma. A product incorporating the Kodama technique has been marketed in Japan; however, the fact that Polymyxin B is strongly nephrotoxic has been a drawback preventing registration in other countries due to the risk of Polymyxin B leaching into blood. Polyethyleneimine as an endotoxin ligand has the twin disadvantages that it strongly adsorbs heparin and also interacts with platelets, leading to coagulation problems in an in vivo application. The potential for adsorbing plasma proteins poses a significant problem for developing an endotoxin adsorbent that is both specific and selective.
European applications (EP 0494848, EP 0129786, Pharmacia Upjohn) disclosed endotoxin removal using an arginine ligand on sepharose. Whereas in vitro trials appeared promising, no further development appears to have been made. Anspach (WO97/33683, DE 19609479) described the immobilization of cationic ligands such as polylysine, N,N-diethylaminoethane, lysine, arginine, histidine or histamine onto polyamide microfiltration membranes and disclosed data on removal of up to about 50% endotoxin from protein solutions. However, applicability was restricted to solutions having a lower protein content than blood or plasma. Hoess, et al. (WO 95/05393) described a peptide having endotoxin adsorbent property. The peptide was composed of hydrophilic, positively charged aminoacids alternated with hydrophobic aminoacids. No data was reported on endotoxin adsorption from blood or plasma. Evans, et al. (WO 96/41185) described immobilizing amidine groups on macroporous beads such that the groups had a specific spacing between the positively charged centers. The material was reportedly suitable for endotoxin removal from plasma and other fluids; however, the material does not appear to be commercially available, Otto, et al. (EP application 0858831) disclosed endotoxin removal from whole blood using albumin as a ligand covalently immobilized onto macroporous polymethyl methacrylate beads. Although in vitro data under static conditions in plasma showed excellent endotoxin adsorption capacity, when applied to whole blood under flowing conditions as in a therapeutic application, the endotoxin removal behavior was very restricted, perhaps due to the weak binding of endotoxin onto immobilized albumin.
Other workers have explored the use of non-selective surfaces for removing endotoxin removal from plasma. Ash, et al. (Biologic DTPF-system TM, ISFA-congress, Saarbrxc3xcken/Germany, Apr. 15-19, 1999) treated endotoxin containing plasma in vitro with fine powdered charcoal, having no ligand, with a surface area of approximately 1000 m2/g charcoal/10 ml plasma. Although high endotoxin removal was reported, the report did not give more details as to what other components had been removed, including beneficial substances. Tetta, et al. (EP 0787500) described the use of positively charged ion-exchange beads for endotoxin removal from plasmas and reported 90% removal in animal trials.
In summary one approach is to remove as much endotoxin as possible simply by using very large adsorbent areas. However, non-specific binding can result in collateral removal of normal blood components such as certain antibodies and coagulation factors. The collateral removal is undesirable. Also, the use of non-specific binding materials is restricted to treatment of plasma, in order to avoid cell activation. Non-specific binding materials are considered impractical for a whole-blood application due to the potential risk of unexpected side reactions.
A different approach is represented by the disclosures of Kodama et al. supra or Otto et al supra based on the use of specific-binding ligands such as polymyxin B or histidine. While such ligands demonstrate sufficient specificity to avoid collateral removal of blood components, the binding capacity is variable across the range of endotoxins likely to be encountered. In order to compensate for low binding capacity a large adsorption chamber might be required, necessitating an unacceptably large extracorporeal blood volume to achieve rapid endotoxin clearance. The lack of binding capacity for a polymyxin B ligand adsorbent was revealed in animal studies in which the adsorbent was only able to clear the endotoxin for a limited time [Otto et al (1997) Therapeutic Apheresis 1:67]. Although the animals lived somewhat longer than untreated controls, the survival rate was not affected.
The use of serum albumin as an adsorbent has been reported. Non-covalent attachment of albumin to bead-type support materials has been reported by Hirae et al. EP 800862, and Suzuki et al EP 028937. Covalently attached albumin has also been disclosed (Otto, EP 848831). The rationale for using albumin is that it already fictions as a binding and transport protein in the bloodstream. The practical use of immobilized albumin is limited by the fact that albumin does not bind endotoxin with sufficient avidity.
Hemodialysis membranes with higher protein adsorption as e.g. AN69 (Gambro-Hospal) are considered to be good surfaces for very low incidence of sepsis related reactions, due to their endotoxin and cytokine adsorption capability.
The problem solved by the present invention is to devise endotoxin adsorption ligands which, on the one hand, have sufficient heterogeneity to effectively adsorb a large variety of endotoxins, while at the same time having sufficient specificity for endotoxins generally to avoid adsorbing other physiologic components of blood, in order not to cause inappropriate side reactions. For this, polycationic species have been synthesized from amino acids which are positively charged at physiological pH of 7.2, e.g. arginine, lysine, or histidine using a polycondensation step in diluted solution in water, such that a very high degree of polydispersity (polycondensation degree, degree of branching, coiling state) results. The broadly distributed oligopeptides can be immobilized on a solid state medium, for example a porous, activated substrate including beads or membranes using conventional coupling reagents such as cyanuric chloride, carbonyldiimidazol, promcyan or water soluble carbodiimide, washed, filled in a housing and sterilized. Surprisingly, the high degree of oligopeptide heterogeneity corresponds to the high degree of heterogeneity of endotoxin resulting in a high capacity for removal of endotoxins from different sources.
Extracorporeal removal of endotoxin from blood of a human or animal subject is accomplished by contacting the blood with an adsorbent composed of a polydisperse oligopeptide of the invention immobilized on a solid state support medium. The support medium is preferably a porous material such as a membrane, particle bed or fiber mat having porosity sufficient to allow passage of blood cells therethrough. Particularly preferred support materials are in the form of beads, which can be filled into a container, the beads having a size sufficient to provide the requisite porosity when packed into a column or filter bed. Examples of suitable bead materials known in the art are provided below.
A device for extracorporeal removal of endotoxin from whole blood can be constructed according to general principles known in the art. The basic components of such a device are a container which is constructed to contain and retain the adsorbent having endotoxin ligand immobilized on a solid phase support medium as described, an inlet and an outlet. The inlet and outlet are positioned with respect to the adsorbent such that blood entering the inlet must contact the adsorbent before exiting through the outlet. Preferably, the geometry of the device is designed to maximize contact of blood with adsorbent during passage through the device. A variety of such designs are known in the art. For example, the device can be a hollow cylinder packed with adsorbent beads, having the inlet at one end and the outlet at the opposite end. Other devices, such as microtubule arrays, can be constructed. All such variations of container geometry and volume and of adsorbent contained therein can be designed according to known principles.
A process for removing endotoxin from the blood of a human or animal subject includes removing a portion of blood from the subject, contacting the blood with an adsorbent according to the invention, whereby the endotoxin binds to the adsorbent, then returning the blood to the subject. Preferably the process is carried out in a continuous flow. The location of the blood vessels of the subject at which blood is removed and returned can be different from one another or the same. In the latter case, single needle techniques are known in the art which reduce the invasiveness of the process.
The duration of treatment will depend upon the endotoxin concentration in the blood, the type of endotoxin present, the capacity of the adsorbent to clear the endotoxin, flow rate and the like, all of which can be monitored and adjusted as is known in the art.
The invention provides broadly distributed: and/or highly branched peptides with a molecular weight range of 500 to 50,000 Da, composed of one or more amino acids which are positively charged at physiological pH (isoelectric point greater than 7.2).